Normal rhythms originate in the sino-atrial node (SAN), a specialized cardiac tissue consisting of only a few thousands pacemaker cells. Malfunction of pacemaker cells due to diseases or aging leads to rhythm generation disorders (e.g., bradycardias) which often necessitate the implantation of electronic pacemakers. Although effective, electronic devices are associated with significant expenses, risks (such as infection, hemorrhage, lung collapse and death) and other disadvantages (e.g., limited battery life, permanent implantation of leads and the lack of autonomic responses). If, encoded by the hyperpolarization-activated cyclic nucleotide-gated or HCN1-4 channel gene family, figures prominently in cardiac pacing. During the last funding period (2004-8), we first obtained insights into the structure-function properties of HCN channels by performing a series of mutagenesis studies. Using the basic knowledge gained and transient adenovirus-mediated gene transfer of the engineered construct HCN1-??? (whose S3-S4 linker has been shortened to favor opening for mimicking the native heteromultimeric If), we subsequently demonstrated that an in vivo HCN-based bioartificial SAN (bio-SAN) can be constructed. To further our effort, here we propose: 1) To test the hypothesis that adeno- associated virus (AAV)-mediated bio-SAN displays a sustained in vivo functional efficacy and safety (24 months) in a swine model of sick sinus syndrome (SSS); 2) To test the hypothesis that right atrial (RA)-converted pacemaker-like cardiomyocytes (CMs) that make up the bio-SAN do not undergo undesirable time-dependent cellular electrophysiological changes in vivo; 3) To test the hypothesis that the HCN-based bio-SAN does not promote reentrant arrhythmias (by high-resolution ex vivo optical mapping), and undergoes time-dependent adaptation in vivo. Taken collectively, the proposed study will shed insights into the basis of cardiac pacing and may lead to biological alternatives or supplement to electronic pacemakers. . PUBLIC HEALTH RELEVANCE: Malfunction of pacemaker cells due to diseases or aging leads to rhythm generation disorders which often necessitate the implantation of electronic pacemakers. Although effective, electronic devices are associated with significant expenses, risks and other disadvantages (e.g., limited battery life, permanent implantation of leads and the lack of autonomic responses). Built on our previous work in the area, the proposed study aims to obtain a better fundamental understanding of the basis of cardiac automaticity and to further develop biological alternatives or supplements to electronic pacemakers with long-term efficacy and safety.